Proteolytically activated receptors (PARs) represent an emerging subset of seven transmembrane G protein-coupled receptors that mediate cell activation events by receptor cleavage at distinct scissile bonds located within receptor amino termini. Differential genomic blotting using a yeast artificial chromosome known to contain the PAR-1 and PAR-2 genes identified the PAR-3 gene within a PAR gene cluster spanning approximately 100 kilobases at 5q13. The PAR-3 gene is relatively small (approximately 12 kilobases); and, like the PAR-1 and PAR-2 genes, it displays a two-exon structure, with the majority of the coding sequence and the proteolytic cleavage site contained within the larger second exon. Sequence analysis of the 5'-flanking region demonstrates that the promoter is TATA-less, similar to that seen with PAR-1, with the identification of nucleic acid motifs potentially involved in transcriptional gene regulation, including AP-1, GATA, and octameric sequences. PAR-3 transcripts were apparent in human vascular endothelial cells, although at considerably lower levels than those of PAR-1 and not significantly modulated by the endothelial cell stimulus tumor necrosis factor-alpha. Likewise, although PAR-3 mRNA was evident in human platelets, receptor cell surface expression was modest (approximately 10%) compared with that of PAR-1. Thus, although PAR-3 is postulated to represent a second thrombin receptor, its modest endothelial cell and platelet expression suggest that PAR-3 activation by alpha-thrombin is less relevant for physiological responses in these mature cells. Rather, given its disparately greater expression in megakaryocytes (and megakaryocyte-like human erythroleukemia cells), a regulatory role in cellular development (by protease activation) could be postulated.